lab diagnosis of syphilis

1. Laboratory Diagnosis Of SyphilisLaboratory Diagnosis Of Syphilis
Dr. Kanwal Deep Singh Lyall
M.D. Microbiology

2. ImportanceImportance

3. Diagnostic Methods Used In SyphilisDiagnostic Methods Used In Syphilis
Demonstration of
Treponemes
Serological tests
1. DGM
2. DFA-TP
3. Silver
impregnation
methods
Non - Treponemal testsNon - Treponemal tests TreponemalTreponemal
CFT
VDRL
RPR, ART
TRUST
USR
Reiter strain Nichol’s strain
RPCF Live T
•TPI
Killed T.
•TPA
•TPIA
•FTA-ABS
Extract of T.
•TPHA
•EIA

4. Specimens CollectedSpecimens Collected
1. Serous fluid from Chancre/ 1° skin lesion
• Collection
2. Blood for serological tests
3. CSF for serological tests

5. Specimen ProcessingSpecimen Processing

6. A.A. Demonstration of TreponemesDemonstration of Treponemes
• Useful in 1° & 2 ° stage of Syphilis
• In congenital Syphilis with superficial skin lesions
1) Dark ground microscopy (motility & morphology)
Merits
rapid technique, useful in 1° & 2 ° stages
Limitations
1. Examine with in 10-30 min
2. Expertise in using DGM
3. Differentiation from commensal spirochetes
4. Low sensitivity (50-80%) – requires 104
/ml for DGM to be +ve

7. Appearance Of T. pallidum Under Dark-fieldAppearance Of T. pallidum Under Dark-field
MicroscopyMicroscopy

8. 2) DFA-TP
• Smears of exudate fixed with acetone → DFA-TP (uses
florescent tagged anti – T. pallidum Ab)
Merits
1. Better & safer, more reliable
2. Specific & sensitive
3. Differentiates between pathogenic & commensal
Treponemes
Demerits
1. Requires specialized equipment & expensive reagents
2. Expertise
3. Can’t diffentiates live & dead spirochetes

9. Reduced

10. A.A. Serological TestsSerological Tests
1) Standard tests for syphilis (STS)
• Or non – specific tests or reagin antibody tests
• Definition : tests for Abs reacting with cardiolipin Ag
• Antigen used – cardiolipin Ag
• Antibody detected – reagin Ab
a) Wassermann CFT (1906)
• Used watery extract of the liver of syphilitic fetus as Ag
b) Kahn flocculation test (1928)
• Used alcoholic extract of ox heart muscle
• Tube flocculation test

11. c) Venereal Disease Research Laboratory (VDRL) USPHS,
New York (1946)
• Definition : microflucculation test which uses cardiolipin Ag to
detect reagin Abs
• Principle : Slide flocculation test
• Procedure :
Inactivate patient’s serum (56° C x 30 min)
Prepare VDRL Ag fresh
Slides with depression (14mm diameter)
0.05ml inactivated serum + 1 drop of VDRL Ag by syringe which
delivers 60 drops/ml
Rotate the slides at 180 rpm x 4 min
Exudate under 10 x objective using 10 x eye piece
Result – reactive / non-reactive/ weak reactive
Quantitation

12. VDRL testVDRL test

13. d) Rapid plasma reagin test ( RPR Test – 1957)
• Finely divided carbon particles are added to cardiolipin
Ag
• Choline chloride
• 100 rpm x 8 min
Advantages over VDRL test
• Unheated serum / plasma can be used
• Visible to naked eye
• Available commercially as a kit
Disadvantages
• Can’t be used with CSF samples

14. RPR testRPR test

15. e) Automated RPR test (ART) – large scale (1968)
f) Automated VDRL – ELISA – IgM / IgG (1987)
g) Toluidine red unheated serum test (TRUST)
• Uses toluidine red particles instead of carbon particles in
TRUST Ag reagent
Advantage
• Can be stored at room temperature ( 26 – 31°C) for ≈ 6
wks
h) USR test
• Unheated serum reagin test which is read
microscopically

16. Interpretation of STSInterpretation of STS
• Positive 3 – 5 wks after infection ( 2 wks after Chancre is
formed)
• 1° stage – 70- 80% reactivity
• 2 ° stage – 100 % (large amount of reagin Abs)
• Latent / late syphilis 60 – 70% reactivity ( Abs reduced
even without treatment
• Following successful treatment reagin Abs fall & become
negative after 6 mnths in 1 syphilis & after 12 – 18 mnths
in 2 syphilis

17. False Positive/ Difficulties In Interpretation Of STSFalse Positive/ Difficulties In Interpretation Of STS
1. Biological false positives (BFP)
• Definition : positive reactions obtained in cardiolipin tests
with negative results in specific treponemal tsets in the
absence of past/ present treponemal infections & not caused
by technical faults
Reason:
• Occurs in about 1% of normal serum
• BFP antibodies are usually IgM type, while reagin Abs are
IgG
Types of BFP
1. Acute BFP
2. Chronic BFP

18. 1. Acute BFP
• Lasts only for a few wks or months
• Acute infection/ injuries/ inflammation
2. Chronic BFP
• Lasts > 6 months
• SLE
• Other CVDs
Conditions associated with BFP
• Leprosy, malaria, hepatitis, RF, IMN, tropical eosinophilia
** non – specific tests → reactive STS → specific T. test
→BFP (< 1/8)

19. 2. Prozone Reaction
• Roughness / abnormal grainy appearance
• Dilutions
3. Incorrect performance of test
• Correct procedure
• Reagents at room temperature
Other sources of error :
Contaminated reagents, haemolysed sample,
lipaemic / contaminated/ incorrect volume of Ag /
incorrect speed of rotation/ incorrect time of rotation
4. Abnormal reactions d/t HIV
• Non – reactive / prozone

20. 2.2. Group specific treponemal testsGroup specific treponemal tests
• Reiter’s treponemes
• Test used – Reiter protein CFT or RPCF test
• Uses – LPS – protein complex Ag
• Sensitivity & specificity < test using T. pallidum
• Some FPs were still observed
• Not in general use now

21. 3.3. Specific treponemal testsSpecific treponemal tests
Uses :
• to confirm reactive STS
• for diagnosis of late / latent syphilis → STS non –
reactive

22. a) Tests using live T. pallidum (Nichol’s strain)
• Test serum is mixed with actively motile Nichol’s
strain
• Incubated anaerobiaclly
• If Abs present in the serum
• Treponems are immobilized
• Observed under DGM
• Results – if > 50% treponems immobilzed = reactive
• If <20% = non – reactive

23. 1. Most specific test but infrequently used now
2. Live treponemes required from infected rabbits
3. Technically difficult to perform

24. b. Tests using killed T. pallidum
i. Treponema pallidum agglutination (TPA)
• Suspension of T. pallidum inactivated by formalin + test
serum → DGM
• Treponemes are agglutinated in the presence of Abs
• Not – very specific
ii. Treponema pallidum immune adherence (TPIA)
• Suspension + test serum + complement + fresh
heparinized blood x incubation
• T. adhere to RBC in presence of Abs

25. iii. Fluorescent treponemal Ab test
• Indirect killed Treponema pallidum → smears fixed
• Add patient’s serum
• Excess serum washed off
• Ab that bind to fixed smear are detected by using
fluorescence labeled anti – human immunoglobulin Abs
• Incubate & wash
• Examine under fluorescent microscope
• If fluorescence present = + ve
• If fluorescence absent = - ve

26. iv. FTA – ABS ( standard reference test)
• Patient’s serum + extract of NP treponemes (Reiter’s
treponemes)
• Removes reagin & group specific Abs
• Rest same as FTA
Advantages
• High specificity & sensitivity
• Can be performed on CSF
• 1st
serological test to become positive
• Modification – IgM FTA – ABS for congenital syphilis
Disadvantages – costly , equipments expensive

27. c. Tests using an extract of T. pallidum
v. Treponema pallidum hemagglutination assay (TPHA)
• Tanned sheep RBCs sensitized with an extract of T. pallidum
• Mixed with patient’s serum
• Clumping of RBC = +ve
• No clumping = - ve
Advantages :
• can be used with CSF sample
• Commercially available kits
• No special equipment required
• Simple to perform & sensitive as FTA –ABS in later stages of
syphilis
Disadvantages : < sensitive than FTA –ABS in 1° syphilis

28. Standard Confirmatory TestStandard Confirmatory Test
• Usually performed in microtitre hemagglutination T.
pallidum (MHA – TP)
• Sera screened in initial dilution of 1/80 but titres ≥
5120 are seen in 2° stages
StageStage VDRL / RPRVDRL / RPR FTA – ABSFTA – ABS TPHATPHA
1° 70 – 80 85 – 100 65 – 85
2° 100 100 100
3° 60 – 70 95 – 100 95 – 100

29. • EIA
• Rapid agglutination assay (latex coated with 3
immunodominant proteins or T. pallidum
• Specificity ≈ TPHA
• Sensitivity > TPHA

30. Non – Specific TestsNon – Specific Tests
VDRL, RPR , TRUST
1. Screening tests
2. Indicate possible active disease
3. Ab titre fall with effective treatment ( 3 mnths interval)
4. May become NR in the late syphilis
5. BFP occur (titre ≤1/8) – FP
6. Prozone reactions – FN

31. Specific Treponemal TestsSpecific Treponemal Tests
TPHA, FTA-ABS
1. Positive test indicates present/past infection
2. High specificity
3. Used to confirm reactive STS
4. +ve for life time → response to treatment
5. Igm & IgG separately
6. New tests → rapid, easy less expensive
x

32. Serological Response At Different Stages OfSerological Response At Different Stages Of
SyphilisSyphilis
1° stage
• One week after chancre – FTA – ABS
• Two weeks later – other specific tests & STS ( 1° & 2° syphilis →
microscopically → 50 -80 % sensitive)
2 ° stage – all serological test are +ve
Latent stage
• Reactivity of STS decrease
• No decrease in STS titres in clinically well person → asymptomatic
neurosyphilis
Late syphilis
• STS → R, WR, NR
• Specific titres → +ve

33. NeurosyphilisNeurosyphilis
• CSF VDRL
• FTA – ABS
• TPHA – most specific

34. Congenital SyphilisCongenital Syphilis
• Detection of IgM Ab in neonate IgM – FTA-ABS/
IgM- EIA/ VDRL-ELISA
• Parallel tests of maternal & neonate sera
• Neonate sera >> mother sera
• Serial testing of neonate serum passively transferred Ab
decrease rapidly & VDRL becomes negative in 3 mnths

35. Immunity in T. pallidum
• Premunition / infection immunity
Treatment
1. Early syphilis - 1°, 2 °, latent infection of ≤2 yrs
• SD injection Benzathine Benzyl Penicillin I/M
• Or tab . Doxycycline 100mg BD orally x 15 days
2. Late syphilis - >2yrs
• Inj. Benzathine Penicillin 24 LU I/M x once weekly x 3
wks
• * Jarish – Herxheemer Reaction
Prophylaxis

36. Non – Venereal TreponemotosesNon – Venereal Treponemotoses
Yaws Pinta Endemic syphilis
Synonyms Frambosia, Pian,
Parangi
Carate, Mal del
pinto
Sibbero – Scotland
Bejel – Middle East
Njovera – Zimbabwe
Dichcgwa – Bechuna
Land
Skerljevo – E. Europe
Siti – Gambia
Geo. Dist Tropical areas of
Africa, Asia,
America
India – Orissa,
AP, MP
Central & S.
America
Causative agent T. Pallidum ss.
Pertenue
T. Pallidum ss.
Carateum
T. Pallidum ss endemicium